Ingénieur(e) de recherche
Faculté des Sciences et Technologies - Nancy
Université de Lorraine
+33 (0)3 72 74 51 90 | firstname.lastname@example.org
Nutrients, 14 (22), pp. 4777.
Allouche, R., Genay, M., Dary-Mourot, A., Hafeez, Z., Miclo, L.
Streptococcus thermophilus, a food grade bacterium, is extensively used in the manufacture of fermented products such as yogurt and cheeses. It has been shown that S. thermophilus strains exhibited varying anti-inflammatory activities in vitro. Our previous study displayed that this activity could be partially due to peptide(s) generated by trypsin hydrolysis of the surface proteins of S. thermophilus LMD-9. Surface protease PrtS could be the source of these peptides during gastrointestinal digestion. Therefore, peptide hydrolysates were obtained by shaving two phenotypically distinct strains of S. thermophilus (LMD-9 PrtS+ and CNRZ-21N PrtS−) with pepsin, a gastric protease, followed or not by trypsinolysis. The peptide hydrolysates of both strains exhibited anti-inflammatory action through the modulation of pro-inflammatory mediators in LPS-stimulated THP-1 macrophages (COX-2, Pro-IL-1β, IL-1β, and IL-8) and LPS-stimulated HT-29 cells (IL-8). Therefore, peptides released from either PrtS+ or PrtS− strains in the gastrointestinal tract during digestion of a product containing this bacterium may display anti-inflammatory effects and reduce the risk of inflammation-related chronic diseases.
Foods, 11 (8), pp. 1157.
Allouche, R., Hafeez, Z., Papier, F., Dary-Mourot, A., Genay, M., Miclo, L.
Nutrients, 14 (11), pp. 2212.
Benoit, S., Chaumontet, C., Violle, N., Boulier, A., Hafeez, Z., Cakir-Kiefer, C., Tomé, D., Schwarz, J., Miclo, L.
Nutrients, 14 (24), pp. 5338.
Pinchaud, K., Hafeez, Z., Auger, S., Chatel, J.-M., Chadi, S., Langella, P., Paoli, J., Dary-Mourot, A., Maguin-Gaté, K., Olivier, J.-L.
Although arachidonic acid (ARA) is the precursor of the majority of eicosanoids, its influence as a food component on health is not well known. Therefore, we investigated its impact on the gut microbiota and gut–brain axis. Groups of male BALB/c mice were fed either a standard diet containing 5% lipids (Std-ARA) or 15%-lipid diets without ARA (HL-ARA) or with 1% ARA (HL + ARA) for 9 weeks. Fatty acid profiles of all three diets were the same. The HL-ARA diet favored the growth of Bifidobacterium pseudolongum contrary to the HL + ARA diet that favored the pro-inflammatory Escherichia–Shigella genus in fecal microbiota. Dietary ARA intake induced 4- and 15-fold colic overexpression of the pro-inflammatory markers IL-1β and CD40, respectively, without affecting those of TNFα and adiponectin. In the brain, dietary ARA intake led to moderate overexpression of GFAP in the hippocampus and cortex. Both the hyperlipidic diets reduced IL-6 and IL-12 in the brain. For the first time, it was shown that dietary ARA altered the gut microbiota, led to low-grade colic inflammation, and induced astrogliosis in the brain. Further work is necessary to determine the involved mechanisms.
Microorganisms, 9 (11), pp. 2380.
Awussi, A. A., Roux, É., Humeau, C., Hafeez, Z., Maigret, B., Chang, O.K., Lecomte, X., Humbert, G., Miclo, L., Genay, M., Perrin, C., Dary-Mourot, A.
Growth of the lactic acid bacterium Streptococcus thermophilus in milk depends on its capacity to hydrolyze proteins of this medium through its surface proteolytic activity. Thus, strains exhibiting the cell envelope proteinase (CEP) PrtS are able to grow in milk at high cellular density. Due to its LPNTG motif, which is possibly the substrate of the sortase A (SrtA), PrtS is anchored to the cell wall in most S. thermophilus strains. Conversely, a soluble extracellular PrtS activity has been reported in the strain 4F44. It corresponds, in fact, to a certain proportion of PrtS that is not anchored to the cell wall but rather is released in the growth medium. The main difference between PrtS of strain 4F44 (PrtS4F44) and other PrtS concerns the absence of a 32-residue imperfect duplication in the prodomain of the CEP, postulated as being required for the maturation and correct subsequent anchoring of PrtS. In fact, both mature (without the prodomain at the N-terminal extremity) and immature (with the prodomain) forms are found in the soluble PrtS4F44 form along with an intact LPNTG at their C-terminal extremity. Investigations we present in this work show that (i) the imperfect duplication is not implied in PrtS maturation; (ii) the maturase PrtM is irrelevant in PrtS maturation which is probably automaturated; and (iii) SrtA allows for the PrtS anchoring in S. thermophilus but the SrtA of strain 4F44 (SrtA4F44) displays an altered activity.
Food & Function, 12, pp. 1415-1431.
Hafeez, Z., Benoit, S., Cakir-Kiefer, C., Dary, A., Miclo, L.
About one in three people are affected by anxiety disorders during their lifetime. Anxiety episodes can be brief due to a stressful event, but anxiety disorders can last at least 6 months. A wide variety of therapeutic drugs is available for the treatment of anxiety disorders, but due to the associated side effects of these anxiolytics, it is interesting to find alternatives. Some food protein hydrolysates or active peptide fragments present in such hydrolysates provide a natural and promising mean for preventing certain forms of anxiety. To date, only a few numbers of hydrolysates or peptides from food proteins with anxiolytic-like activity have been characterized. Most of these hydrolysates or peptides have displayed potent anxiolytic profiles in animal or clinical studies. The results suggest that these molecules may exert their effects at different levels. This paper reviews data of the structure/activity relationship of anxiolytic peptides, their physiological effects displayed in in vitro and in vivo assays, bioavailability, and safety profiles.
Microorganisms, 9 (6), pp. 1113-1113.
Uriot, O., Kebouchi, M., Lorson-Dalibard, É., Galia, W., Denis, S., Chalançon, S., Hafeez, Z., Roux, É., Genay, M., Blanquet-Diot, S., Dary, A.
Despite promising health effects, the probiotic status of Streptococcus thermophilus, a lactic
acid bacterium widely used in dairy industry, requires further documentation of its physiological
status during human gastrointestinal passage. This study aimed to apply recombinant-based in vivo
technology (R-IVET) to identify genes triggered in a S. thermophilus LMD-9 reference strain under
simulated digestive conditions. First, the R-IVET chromosomal cassette and plasmid genomic library
were designed to positively select activated genes. Second, recombinant clones were introduced
into complementary models mimicking the human gut, the Netherlands Organization for Applied
Scientific Research (TNO) gastrointestinal model imitating the human stomach and small intestine,
the Caco-2 TC7 cell line as a model of intestinal epithelium, and anaerobic batch cultures of human
feces as a colon model. All inserts of activated clones displayed a promoter activity that differed
from one digestive condition to another. Our results also showed that S. thermophilus adapted its
metabolism to stressful conditions found in the gastric and colonic competitive environment and
modified its surface proteins during adhesion to Caco-2 TC7 cells. Activated genes were investigated
in a collection of S. thermophilus strains showing various resistance levels to gastrointestinal stresses,
a first stage in the identification of gut resistance markers and a key step in probiotic selection.
Food Research International, 131 (x), pp. 108906-108906.
Kebouchi, M., Hafeez, Z., Le Roux, Y., Dary, A., Genay, M.
The mucus, mainly composed of the glycoproteins mucins, is a rheological substance that covers the intestinal epithelium and acts as a protective barrier against a variety of harmful molecules, microbial infection and varying lumen environment conditions. Alterations in the composition or structure of the mucus could lead to various diseases such as inflammatory bowel disease or colorectal cancer. Recent studies revealed that an exogenous intake of probiotic bacteria or other dietary components (such as bioactive peptides and probiotics) derived from food influence mucus layer properties as well as modulate gene expression and secretion of mucins. Therefore, the use of such components for designing new functional ingredients and then foods, could constitute a novel approach to preserve the properties of mucus. After presenting some aspects of the mucus and mucins in the gastrointestinal tract as well as mucus role in the gut health, this review will address role of dietary ingredients in improving mucus/mucin production and provides new suggestions for further investigations of how dietary ingredients/probiotics based functional foods can be developed to maintain or improve the gut health.
Journal of Dairy Science, 102 (1), pp. 113-123.
Hafeez, Z., Cakir-Kiefer, C., Lecomte, X., Miclo, L., Dary, A.
This study addresses the hypothesis that the extracellular cell-associated X-prolyl dipeptidyl-peptidase activity initially described in Streptococcus thermophilus could be attributable to the intracellular X-prolyl dipeptidyl-peptidase PepX. For this purpose, a PepX-negative mutant of S. thermophilus LMD-9 was constructed by interrupting the pepX gene and named LMD-9-delta-pepX. When cultivated, the S. thermophilus LMD-9 wild type strain grew more rapidly than its delta-pepX mutant counterpart. Thus, the growth rate of the LMD-9-delta-pepX strain was reduced by a factor of 1.5 and 1.6 in milk and LM17 medium (M17 medium supplemented with 2% lactose), respectively. The negative effect of the PepX inactivation on the hydrolysis of β-casomorphin-7 was also observed. Indeed, when incubated with this peptide, the LMD-9-delta-pepX mutant cells were unable to hydrolyze it, whereas this peptide was completely degraded by the S. thermophilus LMD-9 wild type cells. This hydrolysis was not due to leakage of intracellular PepX, as no peptide hydrolysis was high-lighted in cell-free filtrate of wild type strain. Therefore, based on these results, it can be presumed that though lacking an export signal, the intracellular PepX might have accessed the β-casomorphin-7 externally, perhaps via its galactose-binding domain-like fold, this domain being known to help enzymes bind to several proteins and substrates. Therefore, the identification of novel distinctive features of the proteolytic system of S. thermophilus will further enhance its credibility as a starter in milk fermentation.
International Journal of Biosciences, 15 (1), pp. 175-184.
Sultana, F., Ahmad, N., Saeed, F., Jalal, F., Hafeez, Z.
Soybean is a good source of dietary proteins associated with numerous nutritional benefits and attenuates metabolic disorders like myocardial infarction (MI). Present study was aimed to explore the role of hydrolysate of extruded soy proteins concentrate (HESPC) intake in mitigation of MI. Isoprenaline induced MI rat groups (3-6) were fed on diet containing 5.07, 10.14, 15.21, 20.30 g proteins from Hydrolysates of extruded soy proteins concentrates (HESPC), respectively. Group 1 (non-induced MI) and group 2 (Isoprenaline induced MI) were fed on casein diet as control. Feed intake, body and heart weight, lactate dehydrogenase (LDH), creatine kinase-MB (CK), troponin, creatinine and urea, alanine aminotransferase (ALT), aspartate aminotransferase (AST), lipid profile, plasma proteins and homocysteine, cardiac and antioxidant enzymes and histopathology analysis were performed. The diet containing HESPC intake significantly changed body and heart weight. Lowest concentrations of LDH, CK-MB and troponin and highest concentration of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were observed in rats group fed on 20.30 g proteins. Blood cells count was significantly decreased on intake of HESPC in all groups. Plasma lipids, proteins, homocysteine, hepatic enzymes showed decreasing trend in rat groups with increasing percentage of HESPC in diet. Histopathological results showed healing of injured heart tissues was significant on HESPC intake. HESPC diet intake can improve biochemical parameters and cardiac tissues health.
Current Drug Targets, 18 (11), pp. 1269-1280.
Niaz, T., Hafeez, Z., Imran, M.
Background: Global death rate due to cardiovascular diseases (CVDs) is highest as compared to other ailments. Principal risk factor associated with CVDs is hypertension. Major classes of current antihypertensive (AHT) therapies include angiotensin converting enzyme inhibitors (ACEI), angiotensin receptor blockers (ARBs) and calcium channel blockers (CCBs). All these antihypertensive therapeutic drugs have low oral bioavailability and can induce upper respiratory tract abstraction, angioedema, reflex tachycardia and extreme hypotensive effect after oral administration which can cause lethal effects in patients with heart diseases.
Objective: Controlled and targeted release by using antihypertensive nano-medicines can provide better solution to overcome above-mentioned side effects.
Results: Scientific evolution towards the development of biopolymer based nano-carrier systems has unlocked new horizons for safe and/or edible nano drug delivery systems. In this article, we have reviewed in detail various mechanisms of AHT drugs, major draw backs associated with current therapeutic strategies, and the advantages of AHT nano-medicines over conventional drugs. Furthermore, recent reports of bio-based nano/micro -carrier systems with different AHT drugs have been analyzed with their key features. In depth review has been presented for chitosan as a potential carrier of AHT drugs due to its distinctive properties comprising muco-adhesive attribute, permeation enhancement as well as its biocompatible and biodegradable nature.
Conclusion: Chitosan based novel AHT nano-ceuticals can improve oral bioavailability, reduce hydrophobicity and increase the plasma half-life of AHT drugs by their sustained release in lower part of the GIT.
Journal of Agricultural and Food Chemistry, 63 (34), pp. 7522-7531.
Hafeez, Z., Cakir-Kiefer, C., Girardet, J.-M., Lecomte, X., Paris, C., Galia, W., Dary, A., Miclo, L.
The influence on the hydrolysis of isracidin of cell-associated extracellular aminopeptidase and X-prolyl dipeptidyl peptidase activities in addition to protease PrtS of Streptococcus thermophilus strains was investigated. S. thermophilus LMD-9 (PrtS+ phenotype) efficiently hydrolyzed the isracidin mainly through the PrtS activity, whereas strain CNRZ1066 (PrtS- phenotype) and two mutant strains LMD-9-?prtS and LMD-9-?prtS-?htrA also displayed substrate hydrolysis, but different from that of the wild type strain LMD-9. Identification by mass spectrometry of breakdown products of isracidin revealed the existence of novel cell-associated extracellular carboxypeptidase and peptidyl dipeptidase activities in all PrtS- strains, besides known cell-associated extracellular aminopeptidase and X-prolyl dipeptidyl peptidase activities. Both aminopeptidase and peptidyl dipeptidase activities were not able to cleave the isracidin at peptide bonds with proline residues. No hydrolysis of isracidin was detected in cell free filtrate for all the strains studied, indicating that no cell lysis had occurred. Taken together, these results suggested the presence of cell-associated extracellular peptidase activities in S. thermophilus strains that could be vital for the growth of PrtS- strains.
Food Research International, 63 (A), pp. 71-80.
Hafeez, Z., Cakir-Kiefer, C., Roux, É., Perrin, C., Miclo, L., Dary, A.
Besides their basic nutritional role, dietary proteins contain bioactive peptides which are encrypted in their sequence and may modulate different body functions such as digestive, cardiovascular, immune and nervous systems, and therefore contribute in maintaining consumer health. Currently, milk proteins are considered to be the major source of bioactive peptides. The occurrence of these peptides has already been reported in fermented milk products such as yogurt, sour milk or kefir and some of them have been shown to confer health benefits. This review focuses on different strategies that could be employed to enhance the production of bioactive peptides from the milk proteins that will be consequently used to functionalize the fermented milk products. Three types of strategies are developed. The first exploits the proteolytic system of lactic acid bacteria (LAB) or food grade enzymes or combination of both to release the functional peptides from the milk proteins directly in the fermented milk products. The second concerns the supplementation of the fermented milk products with the bioactive peptides obtained outside of the product through the hydrolysis of the purified proteins by the same enzyme sources. Finally, the last consists in the production of the bioactive peptides, initially identified from the milk-proteins, by microorganisms using recombinant DNA technology.
Applied Microbiology and Biotechnology, 97 (22), pp. 9787-9799.
Hafeez, Z., Cakir-Kiefer, C., Girardet, J.-M., Jardin, J., Perrin, C., Dary, A., Miclo, L.
The trend to confer new functional properties to fermented dairy products by supplementation with bioactive peptides is growing in order to encounter the challenge of health-promoting foods. But these functional ingredients have not to be hydrolysed by proteases of bacteria used in the manufacture of these products. One of the two yoghurt bacteria, Streptococcus thermophilus, has long been considered as weakly proteolytic since its only cell wall-associated subtilisin-like protease, called PrtS, is not always present. Nevertheless, a recent study pointed out a possible peptidase activity in certain strains. In this present study, the stability of milk-derived bioactive peptides, e.g. the anxiolytic peptide, αs1-CN-(f91-97), in the presence of two different S. thermophilus strains with PrtS+ or PrtS− phenotype was studied. Both strains appeared to be capable of hydrolysing the αs1-CN-(f91-97) and other bioactive peptides by recurrent removal of N-terminal residues. The hydrolysis was neither due to intracellular peptidases nor to HtrA protease. Results obtained showed that the observed activity originates from the presence at the surface of both strains of an extracellular aminopeptidase activity. Moreover, a cell wall-associated X-prolyl dipeptidyl peptidase activity was also highlighted when β-casomorphin-7 was used as substrate. All of these findings suggest that, in order to use fermented milks as vector of bioactive peptides, the stability of these bioactive peptides in this kind of products implies to carefully characterize the potential action of the surface proteolytic enzymes of S. thermophilus.
5th Edition of Innovations in Food Science and Human Nutrition (IFHN-2022), 20-21 septembre, Barcelone, Espagne
Allouche, R., Hafeez, Z., Dary-Mourot, A., Genay, M., Miclo, L.
23ème Colloque du Club des Bactéries Lactiques, 08-10 juin, Rennes, France
Allouche, R., Hafeez, Z., Papier, F., Dary-Mourot, A., Genay, M., Miclo, L.
Journée doctorale virtuelle franco-allemande et transfrontalière : Biotechnologies et Sciences de la Vie, 10 novembre, dématérialisée
Allouche, R., Hafeez, Z., Dary-Mourot, A., Genay, M., Miclo, L.
Séminaire de l'École Doctorale SIReNa, 13 février, Nancy, France
Allouche, R., Hafeez, Z., Dary, A., Genay, M., Miclo, L.
Streptococcus thermophilus is widely used as a starter culture in the dairy industry and has been awarded generally recognized as safe status (GRAS) by the American Food and Drug Administration. Some strains of S. thermophilus display an anti-inflammatory activity in vitro (Junjua et al., 2016). Inflammation is a part of the regular host reaction to injury or infection caused by pathogens, damaged cells, irritants and allergens. However, the mechanism of action by which this bacterium modulates inflammatory response remains unclear. It has been shown that the hydrolysis of food proteins or endogenous proteins by some digestive proteases releases peptides with various biological activities. Such peptides can also be generated by the surface proteolytic system of Lactic Acid Bacteria (Hafeez et al., 2014) as S. thermophilus, which produces bioactive peptides from bovine caseins (Miclo et al., 2012). These peptides are inactive within the sequence of the parent protein and display their activity after a hydrolysis step. Thus, the assumption that peptides generated in the gastro-intestinal tract from hydrolysis of S. thermophilus surface or intracellular proteins could display an anti?inflammatory activity and contribute to the overall anti-inflammatory effect of the bacterium can be made. Therefore, it is interesting to explore the role of such peptides in the modulation of inflammation. In a first approach, this study aims to investigate the anti-inflammatory properties of hydrolysates genrated after hydrolysis by gastrointestinal enzymes of surface proteins of S. thermophilus. The method involves the recovery of bacterial surface polypeptides by shaving with pepsin. Supernatant obtained after shaving was analysed by RP-HPLC and showed the release of peptides. The next challenge constitutes evaluation in in vitro cell model of anti-inflammatory activity of the peptides obtained and the characterisation of these peptides by mass spectrometry. This study will lead to novel insights into the modulation of host inflammatory response through probable action of peptides obtained from S. thermophilus.
43ème Colloque de la Société de Neuroendocrinologie, 2-4 octobre, Tours, France
Pinchaud, K., Hafeez, Z., Chatel, J.-M., Chadi, S., Auger, S., Dary, A., Maguin Gaté, K., Olivier, J.-L.
L’acide arachidonique (ARA) est le second acide gras polyinsaturé dans le cerveau. L’apport d’ARA est associé à la consommation d’aliments d’origine animale qui semble sous-estimée dans les régimes occidentaux. Une précédente étude de notre laboratoire a montré qu’un régime enrichi en ARA à hauteur de 1% augmente la sensibilité des souris BalB/C males à la neurotoxicité des oligomères de peptides β-amyloïdes, considérés comme étant un des principaux acteurs de la maladie d’Alzheimer. L’objectif de cette étude est d’évaluer l’impact d’un régime enrichi en ARA sur les fonctions cérébrales au travers de modifications du microbiote intestinal et d’altération des communications intestin-cerveau. Pour cela, deux groupes de souris BalB/C ont été nourries avec un régime moyennement hyperlipidique HL-ARA (15% de lipides non enrichi en ARA) ou HL+ARA (15% de lipides et enrichi en ARA à hauteur de 1%) ou encore Std-ARA 5% de lipides non enrichi en ARA) durant 8 semaines complètes. Notre étude n’a montré aucune différence significative concernant le poids des animaux durant l’expérimentation dans chacun des groupes, excepté une augmentation du poids du tissu adipeux mésentérique pour le groupe HL-ARA. Une augmentation du groupe de Bifidobacteriaceae (anti-inflammatoires) dans le microbiote intestinal des souris nourries avec le régime HL-ARA a été mise en évidence comparé au groupe Std-ARA. Cette augmentation de Bifidobacteriaceae est corrélée avec une plus forte présence de lipides dans le régime mais cet effet est renversé par l’ajout d’ARA dans le régime. Aucune modification des marqueurs de l’inflammation n’a été détecté dans le plasma et les fèces de chacun des groupes de souris, mais une augmentation du marqueur des astrocytes GFAP a été observée dans l’hippocampe des souris nourries avec le régime HL+ARA. Il serait intéressant d’étudier les altérations de communication entre l’intestin et cerveau incluant les signaux neuroendocriniens afin de comprendre la cascade impliquée dans la modulation des fonctions cérébrales par l’intestin.
Arachidonic acid (ARA) is the second polyunsaturated fatty acid in the brain. ARA intake is associated with consumption of animal origin products and seems to be underestimated in western diet. A previous study in our laboratory showed that a diet containing 1% ARA increased the sensitivity of male Balb/C mice to the neurotoxicity of the amyloid-β peptide oligomers, considered as the main Alzheimer’s disease agents. The objective of this study was to evaluate the impact of dietary ARA intake on brain functions through gut microbiota modifications and alteration of gut-brain communications. For this, two groups of male BalB/C mice were orally fed with moderately high fat diet, i.e., HL-ARA (15% lipid without ARA) and HL+ARA (15% lipid with 1% ARA) and the third group was fed on standard diet (Std-ARA, 5% lipids without ARA) during 9 weeks. No significant difference was observed in weight gain among the 3 groups except an increase in mesenteric fat tissue in HL-ARA diet group. An increase in Bifidobacteriaceae group (potentially anti-inflammatory) in gut microbiota of HL-ARA diet group was noted compared to standard diet group. This increase in Bifidobacteriaceae was correlated to high lipid contents in diet but this effect was reversed in HL+ARA diet group. No modifications in inflammatory markers were highlighted in plasma and feces samples of the three groups. Contrariwise, higher expression levels of the Glial fibrillary acidic protein were observed in hippocampus of HL+ARA group. It could be interesting to further investigate alterations of the gut-brain communications including neuroendocrine signals.
22nd International Conference of Functional Food Center (FFC) - 10th International Symposium of Academic Society for Functional Foods and Bioactive Compounds (ASFFBC) at Harvard Medical School, 22-23 septembre, Boston, États-Unis
Hafeez, Z., Perrin, C., Dary, A., Chevalot, I., Kapel, R., Chatel, J.M., Miclo, L.
Inflammation, a basic host defensive response, is crucial for resistance to injury, infectious agents or other noxious stimuli. Nevertheless, excessive and persistent inflammation often leads to various chronic diseases such as cardiovascular, osteoporosis, diabetes, obesity and gastro-intestinal inflammatory diseases. According to WHO, chronic diseases are the leading cause of morbidity and mortality both in developed and developing countries, and represent 60% of all deaths in the world. This figure rises to 87% in Europe and it is expected that more people will be affected by chronic diseases over the next few decades. Increased treatment-related health care costs have made chronic diseases a real health problem to the societies and the concern to prevent or treat these illnesses through diet has increased.
Daily diet is comprised of variety of nutrients including proteins. Bioactive peptides derived from dietary proteins particularly milk may modulate different body functions both at intestinal and systemic levels and ultimately contribute in maintaining consumer health. It has been shown that casein hydrolysates or peptides present in them exhibit anti-inflammatory activity by inhibiting and/or reducing the expression of inflammatory markers and/or by modulating their activity. For example, besides the fragment 106-169 of the bovine κ-casein, anti-inflammatory activity was demonstrated for 84VPP86 and 74IPP76 peptides from the bovine β-casein which reduce in vivo the mRNA expression of inflammatory cytokines (IL-6 and IL-1β). Three ways for releasing peptides from native proteins can be: (i) in vitro enzymatic hydrolysis, (ii) during gastrointestinal digestion or (iii) during fermentation by lactic acid bacteria.
Streptococcus thermophilus, a lactic acid bacterium previously known for conferring organoleptic properties to dairy products, has also been shown to generate bioactive peptides from milk proteins through its cell envelope proteinase (PrtS), which is anchored to cell wall by the transpeptidase sortase SrtA. However, during fermentation the pH decreases due to production of lactic acid and consequently the activity of PrtS is reduced or inhibited (optimal activity at pH 7.5), resulting in lower peptide contents. Therefore, to overcome this problem, S. thermophilus LMD-9-delta-srtA mutant strain was constructed to release in growth medium PrtS, which after purification was used to hydrolyze a caseinate to produce a hydrolysate with higher peptide content, which was afterward fractionated.
The main goal of the study was to evaluate the immunomodulatory potential of the peptide fractions obtained after fractionation by ultrafiltration and diafiltration of a hydrolysate resulting from the hydrolysis of a caseinate by PrtS purified from the growth culture of S. thermophilus LMD-9-delta-srtA strain, using peripheral blood mononuclear cells (PBMC) from 4 human donors.
Materials and Methods:
S. thermophilus LMD-9-delta-srtA strain was cultured for 8 h in yeast-lactose (YL) medium to obtain PrtS-rich supernatant. Followed by batch chromatography using diethylaminoethyl (DEAE) cellulose DE23 (Pharmacia, Uppsala, Sweden) resin and discontinuous gradient of NaCl, PrtS-rich fraction was concentrated by ultrafiltration using Amicon® Ultrafiltration system (cutoff threshold 50 kDa, Milipore, Jaffery, USA). The concentrated PrtS-rich fraction was then used to hydrolyze an industrial caseinate comprising of 87% proteins (92% casein and 8% whey proteins). This initial hydrolyzate (F1) was further fractionated by ultrafiltration using a 3 kDa cut-off membrane to isolate non-hydrolysed proteins in the form of retentate (F2) and peptide fraction as permeate (F3). The F3 fraction was then concentrated by ultrafiltration and diafiltration using a 1 kDa cutoff membrane to obtain retentate (F4) and permeate (F5). The peptide fractions were co-incubated in vitro with PBMCs (Clinisciences, Nanterre, France) and secretion of the IL-10 anti-inflammatory cytokine and of the IL-12 pro-inflammatory cytokine was measured in the cellular medium. The IL-10/IL-12 ratio makes it possible to evaluate the immunomodulatory potential of the peptide fractions.
Using DEAE cellulose DE23 batch chromatography with NaCl gradient, the PrtS was predominantly recovered in the fraction eluted by 0.4 M NaCl since about 90% of the initial activity was found in this fraction. Hydrolysis of caseinate by PrtS was not total since size-exclusion chromatography showed that a significant proportion of proteins remained unhydrolyzed under the conditions used. The protein concentration of the hydrolyzate was about 5 g/L with about 1.6 g/L of peptides. F4 fraction containing peptides whose molecular mass was between 1000 and 3000 Da was free of any salts contrary to F3 and F5 fractions. Anti-inflammatory activity of the five peptide fractions was evaluated by quantifying IL-10 and IL-12 production in vitro after co-incubating individually with PBMCs obtained from four healthy donors who did not use anti-inflammatory drugs for a significant period of time. All the fractions tested at concentrations of 0.2 and 1.0 mg of protein matter / mL led to a secretion of IL-10 by PBMCs except F3 and F5 where the secretion of IL-10 was observed only for concentration of 0.2 mg/mL. The absence of secretion with these fractions tested at 1.0 mg/mL was probably due to an excess of salt content. However, F4 fraction induced higher level of IL-10 production (between 70 and 400 pg/mL according to the PBMC donor) followed by F2 fraction regardless of concentration used. No IL-10 was detected when PBS alone was applied. Similarly, none of the peptide fraction favored the release of the pro-inflammatory cytokine IL-12. Results obtained with the cells of all the donors gave the same conclusions.
PBMC treated with F4 fraction, among all peptide fractions, secreted higher levels of IL-10 in vitro and, therefore, this fraction displayed potential anti-inflammatory activity, as it did not trigger any secretion of IL-12. This peptide fraction is rich in peptides with molecular mass ranging between 1 and 3 kDa. Hence, milk proteins represent a promising source of peptides with potential anti-inflammatory activity and these peptides could be released directly by S. thermophilus or by its protease used as a biotechnological tool. It could be interesting to identify the peptide sequences of the F4 fraction in order to characterize it with the aim of using it in the development of functional fermented milk products.
16th World Congress of Food Science and Technology (IUFoST) & XVII Latin American Seminar of Food Science and Technology (ALACCTA), 5-9 août 2012, Foz do Iguaçu, Brésil
Hafeez, Z., Cakir-Kiefer, C., Girardet, J.-M., Dary, A., Miclo, L.
Traditional foods can be functionalized by bioactive peptides either by natural fermentation or ripening and/or by their addition in the form of food ingredients. Milk proteins had been shown as a main source of bioactive peptides. Alpha-casozepine, alpha-s1-CN(f91-100), is a bioactive peptide released by tryptic hydrolysis of bovine alpha-s1-casein that exhibits in vivo anxiolytic activity in three different behavioral tests in rats. The hydrolysate containing this peptide proved effective in clinical studies. In vitro hydrolysis of alpha-casozepine with proteolytic enzymes such as pepsin and chymotrypsin released N-terminal shorter fragment alpha-s1-CN-(f91-97) named heptapeptide that also possesses the same activity. Currently, it has been shown that various Streptococcus thermophilus strains were able to hydrolyze alpha-s1-, alpha-s2- and beta-casein with different efficiency and to release many bioactive peptides but neither alpha-casozepine nor heptapeptide was liberated by the 30 strains of our collection tested. Therefore, to develop a functional food containing such peptides, they could be added directly in dairy products (e.g. fermented milks) and should be resistant to degradation by the proteolytic system of S. thermophilus. The experiments revealed that many bioactive peptides including the heptapeptide were degraded by different S. thermophilus strains even when the strains were lacking the cell surface-associated protease PrtS. Indeed, cell surface-associated aminopeptidase and X-prolyl-dipeptidyl aminopeptidase activities were detected. Therefore, to overcome this problem, we are developing a strategy in which we clone a multicopy of anxiolytic heptapeptide in a plasmid and introduce it into bacteria to produce this peptide as a multimer directly in food product.
18e Colloque du Club des Bactéries Lactiques, 22-24 mai, Clermont-Ferrand, France
Hafeez, Z., Cakir-Kiefer, C., Girardet, J.-M., Dary, A., Miclo, L.
In the last few decades, increased consumers awareness about the health benefits of food products has encouraged the development of various health promoting functional foods. These foods not only contribute to the nutritional requirements but also prevent certain diseases, in order to improve the physical and mental well-being of consumers. In some cases, a bioactive peptide of food origin constitutes the active component of these products. Milk proteins are known to be a major source of bioactive peptides that may impart extra-nutritional benefits. These peptides are encrypted in the sequence of caseins and are released only after protein proteolysis in vitro or by gastric or pancreatic enzymes during the digestion of proteins in vivo or by the enzymes of lactic acid bacteria during manufacture of fermented dairy products. S. thermophilus, that possesses its own proteolytic system, is one of the most widely used lactic acid bacteria in the manufacture of fermented dairy products (yoghurt, cheese). The proteolytic system of S. thermophilus is comprised of cell surface proteinase, peptide transport system and a pool of intracellular peptidases. Thus, the question arises whether bioactive peptides added in the fermented dairy products could remain intact or not in presence of this bacterium. To answer this question, two S. thermophilus strains differing in their cell surface proteolytic activity were evaluated: strain LMD9 which possesses cell envelope proteinase activity (PrtS+) and strain CNRZ1066 without this activity (PrtS-). We studied the resistance of some milk protein derived peptides with different biological activities to S. thermophilus cell wall proteolytic system. We then incubated the peptides, namely anxiolytic [YLGYLEQ - alpha-s1-CN-(f91-97)], antihypertensive [TTMPLW - alpha-s1-CN-(f194-199), FALPQYLK - alpha-s2-CN-(f174-181)] and opioid peptide (YPFPGPI - beta-casomorphin-7, RYLGYLE - alpha-s1-CN-(f90-96)] with the non proliferating cells of LMD-9 and CNRZ1066 strains in phosphate / acetate buffer (12.5 mM, pH 6.5). Surprisingly, two different cell wall-anchored peptidase activities were highlighted. The peptides YLGYLEQ, TTMPLW, RYLGYLE, and FALPQYLK were hydrolyzed by both the strains in a similar way. Liquid chromatography coupled to mass spectrometry analysis revealed that breakdown fragments were generated after successive cleavages of amino acids from the N-terminal of these peptides which provides an evidence of an aminopeptidase activity. However, in case of beta-casomorphin-7, an X-prolyl dipeptidyl aminopeptidase activity was observed which is involved in removal of N-terminal dipeptides containing N-terminal prolyl residue. All the peptides remained stable upon incubation in cell free filtrate of these strains which showed that no intracellular peptidases were released and thus involved in the proteolysis process. Therefore, these peptidase activities may help in optimal bacterial growth which consequently increases the milk fermentation rate but at the same time may also cause degradation of bioactive peptides present or added in the fermented milk products. Hence, screening a strain of S. thermophilus having reduced cell wall proteolytic activity would be required for the production of functional fermented dairy products.
Actes du séminaire de l'École Doctorale RP2E, 19 janvier, Nancy, France
Hafeez, Z., Miclo, L., Girardet, J.-M., Dary, A., Cakir-Kiefer, C.
Actes du séminaire de l'École Doctorale RP2E, 19 janvier, Nancy, France
Hafeez, Z., Miclo, L., Girardet, J.-M., Dary, A., Cakir-Kiefer, C.
17e Colloque du Club des Bactéries Lactiques (CBL), 27-29 octobre, Nancy, France
Hafeez, Z., Cakir-Kiefer, C., Perrin, C., Dary, A., Miclo, L.
Thèse de l'Université de Lorraine, soutenue le 9 décembre 2013